Extracellular guanosine, like adenosine, has been shown to have a plurality of physiological effects both in vitro and in vivo. It affects the growth, differentiation and survival of various cells (Di Iorio P, Benfenati et al. 2004, 2006; Ballerini et al., 2006; Tavares et al., 2005; Molz et al., 2005, Rathbone et al., 2008). Guanine-based purinergic signalling has been particularly investigated in the nervous system. For example, exogenously added guanosine stimulates the division of certain cells in culture including astrocytes, fibroblasts and certain tumour cells, including brain tumour cells (Rathbone et al., 1990; Kim et al., 1991; Ciccarelli et al., 2000; Su et al., 2009; 2010; 2013; Jiang et al., 2006; Rathbone et al. NNNA 2008). It promotes differentiation of fetal neurons (Rathbone et al., 1999; 2008) and PC12 cells, stimulates outgrowth of nerve processes (neurites) (Gysbers and Rathbone, 1992; 1996; Bau et al., 2005; Di Iorio et al., 2002). Guanosine also prevents apoptosis in astrocytes induced by several stimuli (Pettifer et al., 2004; 2007; Jiang et al., 2007; Su et al., 2009). Furthermore, Guanosine has been shown to protect the CNS from insults such as hypoxia-ischemia (Moretto et al., 2009; Thauerer et al., 2012; Thomazi et al., 2008; zur Nedden et al., 2008), stroke (Chang et al., 2008; Connell et al., 2013; Rathbone et al., 2011), spinal cord injury (Jiang et al., 2008a; Jiang et al., 2007; Jiang et al., 2003a; Sam, 2004), and seizure (Schmidt et al., 2005; Schmidt et al., 2000; Soares et al., 2004), and Parkinson's Disease (Giuliani et al., 2012; Su et al., 2009).
Extracellular guanosine is known to stimulate the synthesis and release of several growth factors from cells, which promotes, for example, astrocyte proliferation (Rathbone et al., 1990; Kim et al., 1991; Ciccarelli et al., 2000), partly by stimulating small numbers of microglia in the astrocyte cultures to produce soluble factors, such as IL-1 (Ciccarelli et al., 2000). Additionally, guanosine promotes the synthesis and release of several potentially neuroprotective trophic factors from a variety of cells, including nerve growth factor (NGF) from astrocytes (Middlemiss et al., 1995) as well as basic fibroblast growth factor (FGF/FGF-2) (Su et al., 2009) and transforming growth factor-□ (TGF-□) (Di Iorio et al., 2001). It has been recently shown that exogenous Guanosine can increase intracellular cyclic GMP concentrations through activation of the enzymes hemeoxygenase-1 and hemeoxygenase-2 (HO-1 and HO-2) (Bau et al., 2005).
In some cases guanosine produces its effects by entering cells and interacting with an NGF-dependent protein kinase (Jiang et al., 2006) and under certain circumstances, guanosine acts synergistically with certain growth factors, such as NGF to produce its effects. Guanosine has also been shown to promote the release of adenosine from cells (Ciccarelli et al., 2000); however, most of the effects of guanosine are different from those of adenosine and cannot be explained by adenosine release (Di Iorio et al., 2002).
To date the mechanism through which guanosine exerts its biological effect remains unclear. Many effects of extracellular purine nucleosides and nucleotides are mediated through G-Protein Coupled purine receptors—‘purinoceptors’ (Burnstock G., 2007) that have common structural features. Preliminary evidence for the existence of a high-affinity binding site, specific for guanosine in rat brain membranes (Traversa U., et al., 2002; Traversa et al., 2003; Volpini et al., 2011), analogous to the adenine receptor (Wengert M et al., 2007) has been obtained. Accordingly, it is believed that guanosine is responsible for the activation of a number of intracellular signalling pathways. These intracellular signalling pathways, for example, result in the elevation of cAMP in rat brain membranes (Traversa et al., 2003) and in primary stem cells (Su et al., 2013), PI3kinase/Akt/PKB and mitogen-activated protein kinase and ERK1/2 phosphorylation which are characteristic responses of activated G-Protein Coupled Receptors (Di Iorio P et al., 2001; Pettifer et al, 2007; Traversa U., et al., 2002; Ballerini et al., 2006; Di Iorio et al., 2004; Giuliani et al., 2012). The addition of exogenous guanosine to cultured mouse primary astrocytes also leads to the elevation of intracellular calcium concentrations, although it is unclear whether this is mediated via the putative Guanosine receptor (Chen et al., 2001). Interestingly, the effects of guanosine on the production of trophic factors by astrocytes and the anti-apoptotic effects of guanosine are sensitive to pertussis toxin, an inhibitor of Gi and Go coupled G-protein Receptors (Fields T A and Casey P J, 1997), and are not inhibited by inhibitors of nucleoside or nucleobase transport, which lends support for the hypothesis that guanosine acts extracellularly. Furthermore, rat brain membranes have been shown to have high affinity cell surface binding sites specific for guanosine»inosine that do not bind adenosine (Traversa U., et al., 2002).
On the basis of pharmacological and molecular cloning studies, the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) has subdivided purinoceptors into two major classes: adenosine (P1) receptors and nucleotide (P2) receptors (Fredholm et al., 2001; Abbracchio and Burnstock, 1994). Four subtypes of adenosine receptors have been identified: A1, A2A, A2B, and A3. Each has a unique tissue distribution, signal transduction mechanism, and ligand affinity. All adenosine receptor subtypes are coupled to heterotrimeric G proteins. Activation of A1 or A3 subtypes inhibits adenylate cyclase activity, whereas activation of A2A and A2B subtypes stimulates adenylate cyclase activity. Additionally, A1and A3 subtypes are coupled to other signal transduction pathways, including phosphoinositol hydrolysis and potassium channels (Ramkuran et al., 1993; Linden, 1991).
In addition to their different effects, these receptor subtypes can also be distinguished by the potency order of a series of agonists and antagonists (Palmer and Stiles, 1995). Adenosine is the preferred endogenous agonist at all adenosine receptors. But the naturally-occurring purine inosine, for which no unique receptor has been identified, binds to, and activates A3 receptors (Jin et al., 1997; Linden et al., 1985), producing immunomodulatory and neuroprotective effects (Gomez and Sitkovsky, 2003; Hasko et al., 2004). A3 receptor subtypes exhibit the lowest degree of amino acid sequence identity either with different species homologues (Palmer and Stiles, 1995; Fredholm et al., 2001) or with other adenosine receptor subtypes, resulting in unique pharmacological properties (Linden et al., 1993).
P2 receptors are further divided into P2X ligand-gated ion channels, which are activated solely by ATP, and G-Protein Coupled P2Y receptors which are activated both by extracellular adenine and uracil nucleotides. Eight P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13, 14) have been cloned from mammalian tissues (Abbracchio 2003, Zhang 2002, North 2002, Fredholm, 1997). P2Y receptor activation stimulates phosphoinositol hydrolysis. Additionally, activation of P2Y12, 13, 14 receptors inhibits adenylyl cyclase whereas P2Y11 activation stimulates this enzyme (von Kugelgen and Wetter, 2000).
P1 and P2Y purinoceptors are integral membrane proteins that belong to the class A, rhodopsin-like, G-Protein Coupled Receptor (GPCR) superfamily. These purinoceptors are predicted to share a conserved molecular architecture consisting of seven hydrophobic transmembrane domains (TMDs), which span the plasma membrane, connected by three intracellular and three extracellular loops (Watson et al., 1994).
It is difficult to extract and crystallise GPCRs. Indeed, only the rhodopsin receptor has been crystallised and studied by X-ray diffraction. However, it has been possible to predict the tertiary structure of other GPCRs based on the atomic co-ordinates of the rhodopsin receptor. Since P1 and P2Y receptors have not been crystallised, an understanding of how purinoceptors bind their cognate ligands is based on modelling studies combined with site-directed mutagenesis. GPCRs have low amino acid sequence homology. However, there are several highly conserved key amino acid residues which may be essential for either the structure or function of the receptors. As in other receptor families, similarity between sequences of the P1 or P2Y receptor family is greatest in the hydrophobic TMDs.
Certain well-conserved amino-acid residues of the P2Y receptor subfamily have been used to identify and clone new P2Y receptors (Chambers et al., 2000; Communi et al., 2001; Joost and Methner et al., 1997; Jiang et al., 1997). In contrast, no orphan receptor related to the P1 receptor subfamily has been identified. However, mutational data have identified some amino acid residues important for ligand binding to adenosine receptors (Fredholm et al., 2001).
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